| L-lysine (HO 2 CCH (NH 2) (CH2)4NH 2), is one of the essential a-amino acids, belonging to the basic amino acid just as L-arginine and L-histidine. It could be helpful for promoting development, immune system and central nervous for human. L-lysine is often added in animal feed to significantly improve amino acids absorption balance and keep the livestock healthy and rapid growth. As the level of L-lysine in grain is quite low and can be destroyed easily in the machining process, it was also called the first limited amino acids. As a food, pharmaceutical and cosmetic industry widely used additive, the demand of L-lysine grows rapidly year by year, although the yield of L-lysine is also growing very fast every year, for example, in 2011, the world wide L-lysine yield reached at 1,650,000 tons, while in 2012, L-lysine used in the food additives was more than 1,570,000 tons.In the past, L-lysine was mainly produced by the chemical synthesis and protein hydrolysis, but these methods were limited for source or high-cost of material, long and complex process and environmental pollution, which were gradually eliminated. With the low-cost, environmental friendly, simple and short process, microbial L-lysine production method has been widely used.The current widely used method for L-lysine production is direct fermentation with waste molasses, hydrolyzed starch, acetic acid and ethanol etc as most commonly used carbon source. Microorganism often used in fermentation contains Corynebacterium glutamicum, Brevibacterium lactofermentum, Brevibacterium flavum and Escherichia coli, which were mainly obtained from random mutation breeding in the 1950s. Since the 1970s, with the development of breeding technology, some mutant strains with multiple genetic traits had been created to make the production of L-lysine improved obviously. However, these genetic selected strains with unclear genetic background and unexpected evolution during the fermentation after years often caused problems in the development of L-lysine production industry, such as unstable genetic characteristics and output of L-lysine, diffficult breeding and preserve of strains. Besides, there are many precursors and complex regulatory mechanisms involved in L-lysine biosynthesis, so it is not easy to select strains with improved yield with traditional genetic technology. Furthermore, sequences of key genes or enzymes, strains and production technologies involved in L-lysine biosynthesis have already been well patented by institutions or companies positioned in Japan, South Korea, and US, which also limited the development of modern L-lysine industry.In recent years, with the development of metabolic engineering, comparative genomics, transcriptomics and synthetic biology and based on the assay of metabolic pathways and regulatory mechanisms for desired chemicals, overexpression of exogenous genes or endogenous genes, attenuation or elimination of competition branches, introduction of regulatory elements and high-throughput selection, many engineered strains for industrial production of some specific desired chemicals have been constructed, such as kinds of amino acids, organic acids, biofuels, terpenoids, and polyhydroxyalkanoate. Although Corynebacterium glutamicum has been applied for many kinds of fermentation, the engineered Escherichia coli strains have already been used more and more widely in large scale of valuable chemicals, for their simple and proven genetic manipulation, easy cultivation, and shorter growth cycle.In consideration of the limiting factors involved in L-lysine biosynthesis pathway in E. coli, to construct an MG1655 which can overproduce and excrete L-lysine, the following genetic manipulations were done:First, cadA and ldcC genes, which encode L-lysine decarboxylase I and II respectively, were knocked out to eliminate the degradation of L-lysine accumulated intracellular. Second, pgi gene, which encodes phosphoglucose isomerase, to provide more NADPH. Third, ptsG gene, which encodes the II BC component of glucose-specific phosphoenolpyruvate: carbohydrate phosphotransferase (PTS) system, to provide more PEP. Fourth, sucC/D genes, which encodes succinyl-CoA synthetase, were knocked out to provide more succinyl-CoA. Then, lysCfbr and dapAfbr, encoding aspartate kinase III and dihydrodipicolinate synthase, respectively, were overexpressed after site-directed mutations to remove the feedback inhibition. Finally, lysA and ddh, encoding diaminopimelate decarboxylase and diaminopi melate dehydrogenase, respectively, were over expressed downstream of lysC and dap A. The resulting L-lysine-synthetic strain WML001 was able to produce 3.89 g/L L-lysine in batch fermentation and was therefore used as base strain for further experiment.As an emerging science, synthetic biology has already produced a new set of tools and methods by integration of molecular biology, genomics, information technology and engineering. Through different combinations of natural and synthetic devices, synthetic biology can redesign, improve or even create organisms with desired functions. Although synthetic biology was original from transgenic technology, it was further improved and developed and can be qualified for the tasks difficult for traditional genetic technologies. Riboswitch, a noncoding cis-regulatory elements positioned in 5’-untranslated sequences of mRNAs, could sense intracellular chemicals with aptamers which could convert metabolic signals into a change of expression of downstream genes by conformational changes. For the unique characteristics in regulating gene expressions, Riboswitches have been more and more wildly used in synthetic biology.The 5’UTR of lysC in E. coli could serve as a genetic element to repress the expression of downstream genes, depending on the intracellular concentration of L-lysine. The L-lysine riboswitch, constructed on pUC19, composes of two elements: the 5’UTR of lysC and a dual selection marker tetA. Overexpression of tetA could confers the hosts tetracycline resistance, while this membrane-bound protein could also renders the hosts more sensitive to toxic salts such as NiCl2. While the hosts with riboswitch could produce more L-lysine, expression of tetA would be repressed to confer the cells survival or higher relative growth rate by reducing the absorption of MCI2. Otherwise, hosts, with low yield of L-lysine, could not survive or grow much slower under selection conditions.In previous studies, many feedback-resistant AKs have already been obtained and patented, which is limitation for further application and modification of these patented enzymes. The reported chimeric aspartate kinase BT, composed of the chimeric unit from the N-terminal catalytic region of B. subtilis AK Ⅱ and the C-terminal region from T. thermophilus, was previously proved to have in vitro activity in the presence of L-lysine; however, preliminary experiment showed that the recombinant E. coli strain harboring the BT can’t accumulate L-lysine in the production condition, indicating that the in vivo enzyme activity was not high enough or still sensitive to other molecules in the host. To improve its in vivo activity under L-lysine production condition, this study evolved the rational designed chimeric aspartate kinase BT after random mutagenesis and selected the evolved BTs with high activities using a synthetic RNA device that can recognize the L-lysine. After three rounds of selection, the top three mutated bts were enriched up to the population 72.5%,62.5%, and 71.4%, respectively, indicated that this RNA device could efficiently enrich desired mutants. In order to characterize each evolved genes at protein level, btl, bt2, and bt3 were cloned to the expression vector pET28a with his-tag and were expressed in recombinant E. coli BL21 (DE3). SDS-page analysis showed that all three evolved BTs were able to be expressed in a large amount with high solubility. The target protein fraction accounted more than 30% of the total proteins. Activity analysis of the purified enzymes indicated that the specific activity of the evolved BT1, BT2, and BT3 increased 77%,149% and 160%, respectively, compared with the wild type BT. The activity of the evolved enzyme BT3 was even 13% higher than the commercial widely-used feedback-resistant AKCfbr from E. coli. The activity of the evolved enzymes were not affected by L-lysine and L-threonine addition under serial concentrations, indicating that these enzymes may have potential in industrial application. In order to investigate if the evolved BTs could lead to the L-lysine production in recombinant E. coli, the evolved bt genes were transformed to the host strain WRSA. As our expected, the strains harboring the evolved genes produce more amount of L-lysine than the positive control with the typical commercial widely-used feed-back resistant gene fysCfbr from E. coli in flask batch cultivation, whereas, the strain harboring the wild type BT could not detect L-lysine in the medium. These results indicate that the L-lysine riboselector could be a powerful tool to easily screen for evolved enzymes with high activity from among numerous potential candidates by specifically sensing the inconspicuous metabolite, such as L-lysine, without a requirement for extra expensive analytical instruments or methods. This device can be also used for screening of other key enzymes involved in L-lysine biosynthesis pathway, such as dihydrodipicolinate synthase.In this study, we firstly constructed a basic L-lysine production strain WML001 to analyze the basic limiting factors involved in L-lysine biosynthesis. This study first introduce the RNA device-L-lysine Riboswitch-to evolve the key enzyme aspartate kinase under selection conditions and obtained expected results. These results could be the basic for L-lysine production industry. |
PDF Full Text Request